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Dr Anja Persson
Bahram Amini (research engineer), Caroline Asplund (research engineer), Marica Ekström (research engineer), Jenny Fall (technichian), Holger Eklund (research engineer), Maja Neiman (technichian) and Anna Sköllermo (research engineer)
Production of recombinant PrEST expression clones including cDNA synthesis, cloning, and plasmid purification. All clones are quality controlled with DNA sequencing.
PrEST regions are first amplified with RT-PCR from a total RNA template pool with the specific oligonucleotide primers designed in the PrEST design module. Four different RNA pools are used, three consisting of total RNA from six individual human tissues, and one comprising total RNA from 10 different cell lines. Amplicons are automatically processed with solid phase restriction, and ligated into the plasmid vector pAff8c (Larsson, M. et al, 2000) where the human gene fragment is fused to a histidine tag and an albumin binding domain. After transformation into E. coli BL21(DE3), inserts are verified by DNA sequencing to omit clones with mutations and approved clones are single cell streaked. Plasmids are collected from all purified clones for deposition in the clone library and glycerol stocks are produced for delivery to the protein factory module.
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